Understanding mechanism and improving efficiency of somatic cell nuclear transfer (SCNT) Abstract Understanding the mechanism of cell fate reprogramming is important for both basic biology and regenerative medicine. Of the currently available reprogramming techniques, somatic cell nuclear transfer (SCNT) is the only one that allows efficient and rapid reprogramming of terminally differentiated cells to the totipotent zygote-like state. Totipotency is the ability of a cell to give rise to an organism and its placental tissues. However, despite more than 50 years of effort since the first successful cloning by SCNT, very little progress has been made in understanding how SCNT reprogramming is achieved. Although ectopic expression of certain pluripotency transcription factors (TFs) can reprogram somatic cells into induced pluripotent stem cells (iPSCs), these cells are not totipotent. Moreover, accumulating evidence suggest that SCNT-mediated reprogramming is mechanistically different from that of transcription factor-based iPSC reprograming. Since maintaining undifferentiated stem cells in a lineage-unrestricted nave state is important for therapeutic purposes, understanding how differentiated somatic cells are reprogrammed into a totipotent state is of both biological and clinical importance. During SCNT-mediated reprogramming, donor cell genomes turn off their cell-type specific transcription programs and adopt a new gene expression profile that mimics that of totipotent zygotes. Our preliminary studies indicate that transcriptional reprogramming of donor cells is accomplished within 12 hours following SCNT, indicating that maternal factors present in oocytes can reset the chromatin state of somatic cells quickly upon nuclear transfer. Building upon this intriguing observation, as well as our recently developed techniques in analyzing chromatin accessibility of mouse zygotes and performing maternal factor depletion, we propose to understand the mechanism of SCNT reprogramming and improve SCNT efficiency with the following specific Aims: 1) Identifying and testing TFs and chromatin remodeling factors required for SCNT reprogramming; 2) Overcoming SCNT embryo developmental defects to increase animal term rate. Completion of the proposed study will not only identify oocyte factors important for SCNT-mediated reprogramming, but also improve the SCNT efficiency to achieve maximum term rate. These achievements will have far-reaching implications in the fields of development, stem cell, germ cell, chromatin biology, and regenerative medicine.